Inhibition Of PAF-induced Platelet Aggregation Research Articles

In vitro anti-atherogenic properties of traditional Greek cheese lipid fractions

Given that platelet activating factor (PAF) is a crucial inflammatory phospholipid mediator that is implicated in the mechanism of atherogenesis, the presence of PAF inhibitors in food reinforces their nutritional value in terms of protection against cardiovascular diseases. The aim of the present study was to evaluate the anti-atherogenic (anti-inflammatory) properties of two different types of Greek cheese: Kefalotyri and Ladotyri. Total lipids (TL) of both types of cheese samples were extracted by the method of Bligh and Dyer and separated into total polar lipids (TPL) and total neutral lipids (TNL) by countercurrent distribution. TPL were further separated by preparative thin-layer chromatography (TLC). TL, TPL, TNL and the obtained polar lipid fractions after TLC separation were tested to determine their biological activity towards atherosclerosis based on the in vitro inhibition of PAF-induced platelet aggregation. Both types of cheese samples exhibited strong biological activity, and their lipids were potent PAF inhibitors. Comparing the two types of cheese samples, Ladotyri cheese polar lipid fractions were found to exhibit stronger inhibitory properties than those of Kefalotyri cheese. The fact that both types of cheese were found to contain PAF inhibitors highlights their nutritional value in terms of cardio-protection.

In vitro effects of vitamin supplements on platelet-activating factor and its metabolism in age-related macular degeneration

Objective: The purpose of our study was to investigate for the first time a series of vitamin supplements used for age-related macular degeneration (AMD) as potential inhibitors of platelet-activating factor (PAF).Materials and methods: Various vitamin supplements were tested in washed rabbit platelets (WRPs), in order to investigate the interaction between vitamin supplements (InShape, Nutrof, Ocuvite, Vitalux) and inhibition of PAF-induced platelet aggregation. Additionally, we examined their ability to affect PAF-metabolism, through their in vitro effect on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH).Results: Nutrof exhibited the strongest anti-PAF activity, while Vitalux was the most potent anti-inflammatory factor.Conclusion: This is the first study to bring in surface potent anti-inflammatory and anti-angiogenic activities of some vitamin supplements used against AMD, through their in vitro anti-PAF effects in WRPs and the rabbit plasma and leukocyte PAF metabolism, suggesting a promising role of vitamin supplements and especially resveratrol, concerning its potent anti-angiogenic activity in AMD.

Efficacy and tolerability of rupatadine at four times the recommended dose against histamine- and platelet-activating factor-induced flare responses andex vivoplatelet aggregation in healthy males

European guidelines recommend increasing H1-antihistamine doses up to fourfold in poorly responding patients with urticaria. To assess the efficacy and tolerability of high-dose rupatadine (40 mg) against platelet-activating factor (PAF)- and histamine-induced flare responses in human skin and to verify its anti-PAF activity by assessing its inhibition of PAF-induced platelet aggregation in the blood of subjects receiving rupatadine 40 mg. In the flare study, six male volunteers received a single dose of rupatadine 40 mg. Flares were induced before dosing and up to 96 h afterwards by intradermal PAF and histamine. In the ex vivo study, four male volunteers received an oral dose of rupatadine 40 mg and blood samples were taken 4 h afterwards. Platelet aggregation was assessed in platelet-rich plasma by incubation for 5 min with PAF. Rupatadine 40 mg reached maximal plasma levels of 15·1 ± 4·4 ng mL⁻¹)1 at 1 h and its metabolite, desloratadine, 5·2 ± 0·9 ng mL⁻¹)1 at 2 h. Neither was detectable at 12 h. Inhibition of histamine- and PAF-induced flares was significant within 2 h, maximal at 6 h (87·8 ± 3·1% and 87·1 ± 2·5% inhibition, respectively, P < 0·0001) and still statistically significant at 72 h. Rupatadine 40 mg inhibited PAF-induced platelet aggregation ex vivo by 82 ± 9% (P = 0·023). A single oral dose of rupatadine 40 mg was well tolerated with mild transient somnolence being reported. A single dose of rupatadine at four times the recommended dose is well tolerated, highly effective for up to 72 h against PAF- and histamine-induced dermal flares and has demonstrable PAF-receptor antagonism ex vivo.

Synthesis of All‐trans Arachidonic Acid and Its Effect on Rabbit Platelet Aggregation.

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Synthesis of all- trans arachidonic acid and its effect on rabbit platelet aggregation

A simple and high-yielding method to convert natural all- cis PUFA derivatives to the corresponding all-trans geometrical isomers is described. The method is based on the thiyl radical-catalyzed cis–trans isomerization. The all-trans isomer of arachidonic acid was found to cause rabbit platelet aggregation at concentrations higher than 0.1 mM and inhibition of PAF-induced platelet aggregation in a concentration dependent manner with an IC 50 in the micromolar range.

Platelet-activating-factor-hydrolyzing phospholipase C in the salivary glands and saliva of the mosquito Culex quinquefasciatus.

A phospholipase C activity specific for platelet-activating factor (PAF), named PAF phosphorylcholine hydrolase, was found in the salivary glands and saliva of the human-feeding mosquito Culex quinquefasciatus. The enzymatic activity was demonstrated by inhibition of PAF-induced platelet aggregation, and by identification of substrate consumption and production of diacyl glyceride by electrospray-ionisation mass spectrometry. The activity has a neutral optimal pH and an apparent molecular mass of 40-50 kDa. Two anthropophilic mosquito species, Aedes aegypti and Anopheles gambiae, do not have this salivary activity. The results are interpreted within the evolutionary context of the genera Culex, Aedes and Anopheles.

Inhibition of rabbit platelet aggregation by nucleoside 5′-alkylphosphates: Correlation with inhibition of agonist-induced calcium influx

We investigated the effects of uridine 5′-alkylphosphates on agonist-induced aggregation, increased intracellular calcium concentration [Ca 2+] i, and Ca 2+ (Mn 2+) influx in washed rabbit platelets. Uridine 5′-hexadecylphosphate (UMPC16) and uridine 5′-eicosylphosphate (UMPC20) at a concentration of 1 × 10 −5 M inhibited platelet aggregation induced by platelet-activating factor (PAF), thrombin, arachidonic acid, and ADP. UMPC16 did not cause significant interference in the binding of [ 3H- acetyl]PAF to platelets. The inhibition of PAF-induced platelet aggregation by UMPC16 was dependent upon the addition time; UMPC16 was ineffective at 60 sec when the extracellular calcium uptake reached the maximum level in PAF-stimulated platelets. Furthermore, UMPC16 inhibited guanosine 5′- O-(3-thiotriphosphate)-induced platelet aggregation but did not affect ionophore A23187- and calcium-independent agonist phorbol 12-myristate 13-acetate-induced platelet aggregation. UMPC16 markedly inhibited the Ca 2+ (Mn 2+) influx induced by PAF and ADP, and partly inhibited the [Ca 2+] i increase induced by the receptor-mediated stimulation. On the other hand, UMPC16 did not affect the [Ca 2+] i increase and Ca 2+ (Mn 2+) influx induced by ionomycin. These experiments suggest that inhibition of calcium influx associated with receptor-mediated platelet activation may be involved in the action of UMPC16.

Separation of biologically active lipids from red wine.

A number of lines of evidence suggest that red wine exerts a protective effect against coronary heart disease, but the nature of the protective compounds is unclear and the mechanism is incompletely understood. In this study, total lipids of a Greek red wine were separated into neutral and polar lipids. Polar lipids were further separated into glyco- and phospholipids, which were fractionated by HPLC. Each lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF) and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. A significant number of glyco- and phospholipids that exerted the above biological activities were detected. Structural data of an active phosphoglycolipid are also provided. trans-Resveratrol demonstrated also a dose-dependent inhibition of PAF-induced platelet aggregation along with the already reported inhibitory activity against thrombin and adenosine-5'-diphosphate. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in red wine may contribute to the protective role of red wine against atherosclerosis.

Synthesis and preliminary pharmacological evaluation of some cytisine derivatives

Thirty-one N-derivatives of cytisine were prepared in order to modify its pharmacological profile and to obtain compounds of potential therapeutic interest either at a peripheral or central level, particularly as nicotinic ligands with improved ability to cross the blood–brain barrier. Actually, with the introduction of different kinds of substituent on the basic nitrogen of cytisine a variety of activities were observed, both in vivo (analgesic, dopamine antagonism, antihypertensive, inhibition of stress-induced ulcers, antiinflammatory, protection from PAF-induced mortality, hypoglycemic) and in vitro (positive cardio–inotropic, β-adrenergic antagonism, α 1- and α 2-antagonism, inhibition of PAF-induced platelet aggregation). Six randomly selected compounds were tested for the ability to recognize a central nicotinic receptor and four of them exhibited K i values in the range 30–163 nM.

Effects of chroman derivatives on platelet aggregation induced by some aggregating agents in rabbits.

1. We synthesized 10 chroman derivatives (CD-1 to CD-10) derived from khellactone, including praeruptorin A and praeruptorin B and examined the effects of these compounds on rabbit platelet aggregation. 2. These compounds exhibited an inhibitory effect on platelet-activating factor (PAF)-induced platelet aggregation and their effects were more potent on PAF-induced platelet aggregation than on adenosine triphosphate (ADP)-, arachidonate (AA)- or collagen-induced platelet aggregation. In particular, (+/-)-cis-5-methoxy-6-methoxycarbonyl-2,2-dimethyl-3,4-ditiglyloxychr oman (CD-6), (+/-)-cis-5-methoxy-6-(2-methoxycarbonylethenyl)-2,2-dimethyl- 3,4-ditiglyloxychroman (CD-8), (+/-)-cis-3,4-diacetoxy-5-methoxy-2,2-dimethyl-6-propylchroman (CD-9) and (+/-)-cis-5-methoxy-2,2-dimethyl-6-propyl-3,4-ditiglyloxychroman (CD-10) showed a moderate inhibition of PAF-induced platelet aggregation. 3. From these findings, it is suggested that compounds with potent PAF antagonist activities have the following features: (i) a tiglyloxy moiety is required at the 3 and 4 positions; and (ii) the methoxy moiety is also required at the 5 position of chroman skeleton in khellactone.

Inhibition by Glaucocalyxin A of Aggregation of Rabbit Platelets Induced by ADP, Arachidonic Acid and Platelet-Activating Factor, and Inhibition of [3H]-PAF Binding

Glaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 mumol/l, 14.1 mumol/l respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 mumol/l. Analysis of [3H]-PAF binding showed that glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 mumol/l, which was consistent with its inhibition of PAF-induced platelet aggregation.

Inhibition of ex-vivo PAF-induced platelet aggregation by the PAF-antagonist RP 48740: relationship to plasma concentrations in healthy volunteers

RP 48740, 3-(3-pyridyl)-1H,3H-pyrrolo [1,2-c] thiazole-7-carboxamide, a specific competitive PAF-receptor antagonist in vitro, was given to 29 healthy male volunteers for 7 days. Plasma drug concentrations and ex-vivo PAF-induced platelet aggregation were assessed on Days 1, 4, and 7. RP 48740 had linear pharmacokinetics after single and repeated doses. It caused stable inhibition of PAF-induced platelet aggregation in a dose-dependent manner. The effect disappeared within 24 h, even after 7 days of repeated doses. The effect of RP 48740 displayed a sigmoidal relation to the plasma drug concentration; I50 2.3 (0.3) mg.l-1. There were no clinical or biological adverse reactions to RP 48740 during the study.

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7–96 μM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.

Platelet-activating factor (PAF) acetylhydrolaseとリポ蛋白

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma was estimated in 35 patients with cerebral thrombosis, 18 with essential hypertension, and 34 healthy adults age-matched with two groups of patients. In all cerebrovascular patients more than 3 months had elapsed since their stroke. The assay measured the inhibition of PAF-induced platelet aggregation by the preincubation of a standard PAF solution with sample plasma. For this purpose, platelet aggregation induced by a PAF solution which had been preincubated with an equal amount of citrated plasma at 37°C for 15min was compared with that induced by a control PAF solution which had been preincubated with phosphate-buffered saline.The average values of plasma PAF acetylhy-drolase activity in the cerebrovascular patients and the hypertensives were 4.18±3.08 and 1.71±0.27μg/ml, respectively. These values were significantly higher than the average value of plasma PAF acetylhydrolase activity in the control group: 1.51±0.51μg/ml. In each of three groups, plasma PAF acetylhydrolase activity correlated positively with the level of serum LDL-cholesterol, reflecting the complex formation of this enzyme with plasma lipoproteins, mainly with LDL. Serum LDL-cholesterol level in the cerebrovascular patients was higher than that in the control, which may account, in part, for the higher-than-normal plasma PAF acetylhydrolase activity in the patients. However, relative activity of PAF acetylhydrolase to LDL-cholesterol level was found to be higher in both patient groups as compared to the control. This result may suggest that patients' LDL contains higher activity of the enzyme than that of healthy subjects, and such a fact could be considered as one of the qualitative alterations of lipoproteins in these patients.

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.

Inhibition of platelet-activating factor by rabbit C-reactive protein

Incubation of 0.5 n M platelet-activating factor (PAF) with 123 μg/ml C-reactive protein (CRP) for 60 min at room temperature in the presence of 2 m M calcium resulted in total inhibition of the platelet-aggregating capacity of 0.5 n M PAF. There was no inhibition of platelet aggregation if PAF and CRP were added to the platelets simultaneously, without prior incubation of the mixture, or if calcium was not present during the incubation of CRP and PAF. The inhibitory effect of CRP (123 μg/ml) was dependent upon the time of incubation with PAF (1 n M). We observed 0, 42, and 85% inhibition of platelet aggregation with incubations of 0, 20, and 120 min, respectively. The inhibitory effect of CRP was also concentration dependent. After incubations of 20 min at room temperature with 0.5 n M PAF there was no inhibition of PAF-induced platelet aggregation using 12.3 μg/ml CRP with the inhibition increasing to 73% using 123 μg/ml CRP. Pretreatment of the platelets with CRP (123 μg/ml) for 60 min before addition of PAF did not affect the induction of platelet aggregation by PAF. These observations indicate that CRP inhibits PAF and strongly suggest that binding of CRP to PAF, presumably to the phosphocholine moiety of PAF, is required for this inhibition.